Polymeric delivery agents and delivery agent compounds

ABSTRACT

Polymeric delivery agents, delivery agent compounds and compositions comprising them which are useful in the delivery of active agents are provided. Methods of administration and preparation are provided as well.

This application is a continuation of U.S. Ser. No. 10/447,608, filedMay 28, 2003, now U.S. Pat. No. 7,208,483, which is a continuation ofU.S. Ser. No. 09/889,005, filed Oct. 9, 2001, now U.S. Pat. No.6,627,228, which is a national phase application of InternationalApplication No. PCT/US00/00476, filed Jan. 7, 2000, which was publishedin English as International Publication No. WO 00/40203 and claims thebenefit of U.S. Provisional Patent Application No. 60/115,273, filedJan. 8, 1999, the entire disclosures of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to compositions for delivering activeagents, and particularly biologically or chemically active agents. Thecompositions comprise a polymeric delivery agent or delivery agentcompound which facilitates the delivery of the active agent to a target.These polymeric delivery agents and delivery agent compounds are wellsuited to form non-covalent mixtures with active agents foradministration to animals. Methods for the preparation and for theadministration of such compositions are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents are often severelylimited by biological, chemical, and physical barriers. Typically, thesebarriers are imposed by the environment through which delivery occurs,the environment of the target for delivery, or the target itself.Biologically or chemically active agents are particularly vulnerable tosuch barriers.

In the delivery to animals of pharmacological and therapeutic agents,barriers are imposed by the body. Physical barriers such as the skin andvarious organ membranes are relatively impermeable to certain activeagents but must be traversed before reaching a target, such as thecirculatory system. Chemical barriers include, but are not limited to,pH variations in the gastrointestinal (GI) tract and degrading enzymes.

Oral delivery of many biologically or chemically active agents would bethe route of choice for administration to animals if not for biological,chemical, and physical barriers. Among the numerous agents which are nottypically amenable to oral administration are biologically or chemicallyactive peptides, such as calcitonin and insulin; polysaccharides, and inparticular mucopolysaccharides including, but not limited to, heparin;heparinoids; antibiotics; and other organic substances. These agents maybe rendered ineffective or may be destroyed in the GI tract by acidhydrolysis, enzymes, or the like, or may simply not be absorbed.

Many delivery agents are fairly hydrophobic, whereas many active agentsare hydrophilic. The differential aqueous solubility between thedelivery agent and the active agent can be problematic in designingcommercially acceptable dosage formulations which exhibit biologicalactivity in vivo. Thus, the ability to alter the solubility of adelivery agent would allow one to tailor the delivery agent to meet theneeds of the cargo in order to optimize its bioavailability.

The pH within the gastrointestinal tract typically ranges from about 1to about 8, while many delivery agents remain soluble over a range ofonly 2-2.5 pH units. During oral delivery, a significant amount of sucha delivery agent could precipitate out in the stomach due to the localacidity. The precipitated delivery agent would then be unavailable fordelivery of active agent to a point further along the GI tract.Increasing the span of pH solubility of the delivery agent would allowmore effective delivery at lower concentrations of delivery agent.

Delivery agents generally tend to self-aggregate into micellular-likestructures. The competition between self association and associationwith the active agent typically results in at least a portion of thedelivery agent being unavailable for effective delivery of the (activeagent. Thus, a corresponding portion of the active agent that wasadministered may not be effectively delivered to the target. Inhibitingself aggregation of the delivery agent would increase the availabilityof delivery agent for delivery of the active agent.

Various delivery agents for oral administration of active agents havebeen developed in recent years. These delivery agents includeproteinoids, modified vegetable proteins, acylated or sulfonated aminoacids, acylated or sulfonated amino acid ketones, and acylated orsulfonated amino acid aldehydes. See, U.S. Pat. Nos. 5,401,516;5,443,841; 5,451,410; 5,541,155; 5,629,020; 5,643,957; 5,693,338;5,709,861; 5,714,167; 5,766,633; 5,773,647; 5,792,451; 5,820,881;5,863,944; 5,866,536; and RE35,862. These delivery agents promotesystemic absorption of active agents in the gastrointestinal tract. Theinteraction between the delivery agent and the active agent, as well asthe interaction between the delivery agent and the cell membrane, may beimportant for absorption. See, U.S. Pat. No. 5,714,167.

There is a need for delivery agents whose solubility can be modified fora particular need, thereby changing the concentration of solubledelivery agent which is available for delivery of an active agent.

Therefore, there is a need for alternate and improved delivery agents.

SUMMARY OF THE INVENTION

The present invention provides polymeric delivery agents which areuseful in the delivery of active agents. The polymeric delivery agentcomprises a polymer conjugated to a modified amino acid or derivativethereof via a linkage group selected from the group consisting of—NHC(O)NH—, —C(O)NH—, —NHC(O)—, —OOC—, —COO—, —NHC(O)O—, —OC(O)NH—,—CH₂NH—, —NHCH₂—, —CH₂NHC(O)O—, —OC(O)NHCH₂—, —CH₂NHCOCH₂O—,—OC(O)NHCH₂—, —NHC(O)CH₂O—, —OCH₂C(O)NH—, —NH—, —O—, and carbon-carbonbond, with the proviso that the polymeric delivery agent is not apolypeptide or polyamino acid. The modified amino acids may be acylatedor sulfonated amino acids, ketones or aldehydes of acylated orsulfonated amino acids, salts thereof, or polyamino acids orpolypeptides of any of the foregoing.

The polymer may be any polymer including, but not limited to,alternating copolymers, block copolymers and random copolymers, whichare safe for use in mammals. Preferred polymers include, but are notlimited to, polyethylene; polyacrylates; polymethacrylates; poly(oxyethylene); poly(propylene); polypropylene glycol; polyethyleneglycol (PEG) and derivatives thereof, such as PEG-maleic anhydridecopolymers; and derivatives and combinations thereof. The molecularweight of the polymer typically ranges from about 100 to abut 200,000daltons. The molecular weight of the polymer preferably ranges fromabout 200 to about 10,000 daltons. In one embodiment, the molecularweight of the polymer ranges from about 200 to about 600 daltons andmore preferably ranges from about 300 to about 550 daltons.

According to one embodiment, the polymeric delivery agent comprisesunits having the formula

or salts thereof where R¹ is a modified amino acid which is bonded tothe polymer via a linkage group selected from the group consisting of—NHC(O)NH—, —C(O)NH—, —NHC(O)—, —OOC—, —COO—, —NHC(O)O—, —CC(O)NH—,—CH₂NH—, —NHCH₂—, —CH₂NHC(O)O—, —OC(O)NHCH₂—, CH₂NHCOCH₂O—,—OC(O)NHCH₂—, —NHC(O)CH₂O—, —OCH₂(O)NH—, —NH—, —O—, and carbon-carbonbond; R² is H or —CH₃; and R¹ is H or —COOH. Preferably, R¹ is —R³-R⁴where R³ is —NHC(O)NH—, —C(O)NH—, —NHC(O)—, —OOC—, —COO—, —NHC(O)O—,—OC(O)NH—, —CH₂NH—, —NHCH₂—, —CH₂NHC(O)O—, —OC(O)NH—, —CH₂NHCOCH₂O—,—OCH₂C(O)NHCH₂—, —NHC(O)CH₂O—, —OCH(O)NH—, —NH—, —O—, or carbon-carbonbond; and R⁴ has the formula

where

R⁵, R⁶, R⁷, R⁸, and R⁹ are independently a bond to R³, or H, Cl, Br, F,—OH, —CH₃, —OCH₃, or —(CH₂)_(m)CH₃;

R¹⁰ is a bond to R³ or —COOH, or —C(O)NH—R¹¹-R¹²;

R¹¹ is a substituted or unsubstituted, linear or branched alkylenehaving a chain length of from about 1 to about 11 or —R¹³-R¹⁴—;

R¹² is a bond to R³ or is —COOH, —NH, —OH, —C(O)—R¹⁵, —COO—R¹⁵, —NHR¹⁵,—OR¹⁵, Cl, or Br;

R¹³ is a substituted or unsubstituted phenylene;

R¹⁴ is a substituted or unsubstituted, linear or branched alkylenehaving a chain length of from about 1 to about 5;

R¹⁵ is a bond to R³; and

m is from about 1 to about 4

Preferably, R⁴ is selected from the group consisting of

and salts thereof.

Preferably, R⁹ is —OCH₃ or —OH. According to a preferred embodiment R¹⁰is —NH—R¹¹-R¹² and R¹¹ is —(CH₂)₇—, —(CH₂)₆—, —(C₆H₅)—(CH₂)₃—,—(C₆H₅)—CH₂—, or —(CH₂)₂—NH—C(O)—CH₂—.

Another embodiment is a polymeric delivery agent having units of theformulaR¹⁶-R²⁴—CH₂CH₂—R¹⁷or salts thereof where R¹⁶ is defined as R¹ above; R¹⁷ is —OH, —OCH₃, or—R¹⁸; R¹⁸ is defined as R¹ above; and R²⁴ is a polymer having units of—(CH₂CH₂O)—, —(CH(CH₃)CH₂O)—, or a combination thereof. R²⁶ typicallycontains from about 3 to about 200 polymeric units. R²⁴ may be a randomcopolymer or a block copolymer R¹⁸ may be the same or different thanR¹⁶.

A preferred embodiment of the polymeric delivery agent has units of theformula

or salts thereof where R¹⁶, R¹⁷, and R¹⁸ are defined as above; R²³ is Hor —CH₃; and n is from about 3 to about 200. Preferably, R¹⁶ and R¹⁶ are—OOC—R⁶. According to a preferred embodiment, R¹⁷ is —OCH₃. According toanother preferred embodiment, R¹⁶ is —NHC(O)—R⁴ or —NHC(O)O—R⁴ and nranges from about 4 to about 15.

Yet another embodiment is a polymeric delivery agent having units of theformula

or salts thereof where R²⁰, R²¹, and R²² independently are H or aredefined as R¹ above; a, b, and c independently are integers from about 1to about 50; and d ranges from about 2 to about 10. Preferably, R²⁰,R²¹, and R²² independently are —COO—R⁴. Preferably, d is about 6.

Examples of polymeric delivery agents and units for the polymericdelivery agents include, but are not limited to,

wherein x is 0.02 to 0.5, preferably 0.05 (Conjugate 1 is a randompolymer);

wherein x is 0.02 to 0.5, preferably 0.06 (Conjugate 1 is a randompolymer);

wherein

k=1-11, preferably 7 or 9

n=10 to 50, preferably 33, and

m=5 to 15, preferably 9.

Conjugate 3 is the above structure where k=7, n=33 and m=8;

I-COO—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OH Conjugate 4.

I-COO—CH₂CH₂O(CH₂CH₂O)₃CH₂CH₂OH Conjugate 5

I-COO—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 6

I-COO—CH₂CH₂O(CH₂CH₂O)₇CH₂CH₂OH Conjugate 7

II-COO—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OH Conjugate 8

III-COO—CH₂CH₂O(CH₂CH₂O)₇CH₂CH₂OH Conjugate 9

I-COO—CH₂CH₂O(CH₂CH₂O)₂₂CH₂CH₂OH Conjugate 10

I-COO—CH₂CH₂O(CH₂CH₂O)₁₀₂CH₂CH₂—OOC—I Conjugate 11

PEG branched (8 arms):

The modified amino acid I-COO is attached at the —OH group through anester linkage at 4 of the 8 “arms”.

-   -   Conjugate 12

I-COO—CH₂CH₂O(CH₂CH₂O)₁₄CH₂CH₂OCH₃ Conjugate 13

IV-COO —CH₂CH₂O(CH₂CH₂O)₄CH₂CH₂OH Conjugate 14

V-COO—CH₂CH₂O(CH₂CH₂O)₄CH₂CH₂OH Conjugate 15

IV-COO—CH₂CH₂O(CH₂CH₂O)₆CH₂CH₂OH Conjugate 16

I-CH₂NH—CO—O—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 17

I-CH₂NH—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 18

II-COO—CH₂CH₂O(CH₂CH₂O)₁₀CH₂CH₂OCH₃ Conjugate 19

II-COO—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 20

I-COO—CH₂CH₂O(CH₂CH₂O)₁₀CH₂CH₂OCH₃ Conjugate 21

I-CH₂NH—CO—CH₂O—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 22

I-CH₂NH—CO—CH₂O—CH₂CH₂O(CH₂CH₂O)₁₀CH₂CH₂OCH₃ Conjugate 23

VI-COO—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 24

VI-COO—CH₂CH₂O(CH₂CH₂O)₁₀CH₂CH₂OCH₃ Conjugate 25

VII-COO—CH₂CH₂O(CH₂CH₂O)₁₀CH₂CH₂OCH₃ Conjugate 26

VIII-COO—CH₂CH₂O(CH₂CH₂O)₆CH₂CH₂OH Conjugate 27

III-COO—CH₂CH₂O(CH₂CH₂O)₄CH₂CH₂OH Conjugate 28

III-COO—CH₂CH₂O(CH₂CH₂O)₆CH₂CH₂OH Conjugate 29

(I-COOH)-5-CH₂NH—CO—CH₂O—CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 30

PEG branched (8 arms):

The modified amino acid I-COO is attached at the —OH group through anester linkage at 6 of the 8 “arms” Conjugate 32

I-COO—CH₂CH₂O—(CH₂CH₂O)₁₁—CH₂CH₂—OOC—I Conjugate 33

(I-COOH)-5-CH₂NH—CO—OCH₂CH₂—(OCH₂CH₂)₄₃—OCH₂CH₂—OCH Conjugate 34

PEG branched (8 arms):

The modified amino acid I-COO is attached at the —OH group through anester linkage at 4 of the 8 “arms”. Conjugate 35The modified amino acid I-COO is attached at the —OH group through anester linkage at 5 of the 8 “arms”. Conjugate 36The modified amino acid I-COO is attached at the —OH group through anester linkage at 7 of the 8 “arms”. Conjugate 37The modified amino acid I-COO is attached at the —OH group through anester linkage at 8 of the 8 “arms”. Conjugate 38

I-COO—CH₂CH₂O—(CH₂CH₂O)₄₅—CH₂CH₂OOC—I Conjugate 39

I-NH—COO—CH₂CH₂O—(CH₂CH₂O)₄₂—CH₂CH₂OCH₃ Conjugate 40

I-COO—CH₂CH₂O—(CH₂CH₂O)₄—CH₂CH₂OOC—I Conjugate 41

XI-COO —CH₂CH₂O—(CH₂CH₂O)₄—CH₂CH₂OH Conjugate 42

X-COO—CH₂CH₂O—(CH₂CH₂O)₁₀—CH₂CH₂OCH₃ Conjugate 43

X-COO—CH₂CH₂O—(CH₂CH₂O)₁₁—CH₂CH₂OH Conjugate 44

X-COO—CH₂CH₂O—(CH₂CH₂O)₂₀—CH₂CH₂O—CO—X Conjugate 45

X-COO—CH₂CH₂O—(CH₂CH₂O)₂₀—CH₂CH₂OH Conjugate 46

X-COO—CH₂CH₂O—(CH₂CH₂O)₁₁—CH₂CH₂O—CO—X Conjugate 47

IX-COO—CH₂CH₂O—(CH₂CH₂O)₅—CH₂CH₂OCH₃ Conjugate 48

The number of polymeric units specified in the aforementioned polymericdelivery are an average number of units. The number of units in thepolymers typically may vary by up to about 10%.

Another embodiment provides a composition comprising (A) at least oneactive agent; and (B) at least one of the aforementioned polymericdelivery agents. The active agent preferably is a biologically orchemically active agent. Methods for the preparation and administrationof the composition are also provided. These compositions are useful inthe delivery of active agents to selected biological systems and forincreasing or improving the bioavailability of the active agent comparedto administration of the active agent without the delivery agent.

The invention also includes a method of preparing a polymeric deliveryagent by conjugating a modified amino acid to a polymer, via one of theaforementioned linkage groups.

The invention further includes delivery agent compounds having theformulae

and salts thereof, including but not limited to sodium salts. Thesedelivery agent compounds are useful for facilitating the delivery of anactive agent. Another embodiment is a composition comprising one of theaforementioned delivery agent compounds and an active agent.

DETAILED DESCRIPTION OF THE INVENTION

These compositions may be used to deliver various active agents throughor across various biological, chemical, and physical barriers and areparticularly suited for delivering active agents that are subject toenvironmental degradation. The compositions of the subject invention areparticularly useful for delivering or administering biologically orchemically active agents to any animals, including but not limited tobirds such as chickens; mammals, such as rodents, cows, pigs, dogs,cats, primates, and particularly humans; and insects.

Other advantages of the present invention include the use ofeasy-to-prepare, inexpensive raw materials. The compositions and themethods of the present invention are cost effective, easy to perform,and amenable to industrial scale up for commercial production.

The presently disclosed compositions deliver active agents, particularlyin oral, intranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular and ocular systemsas well as traversing the blood-brain barrier. Coadministration of anactive agent and a polymer-delivery agent conjugate results in anincreased bioavailability of the active agent compared to administrationof the active agent alone.

The term “salts” as used in this application includes but are notlimited to organic and inorganic salts, for example alkali-metal salts,such as sodium, potassium and lithium; alkaline-earth metal salts, suchas magnesium, calcium or barium; ammonium salts; basic amino acids suchas lysine or arginine; and organic amines, such as dimethylamine orpyridine. Preferably, the salts are sodium salts.

Active Agents

Active agents suitable for use in the present invention includebiologically active agents, and chemically active agents, including, butnot limited to, pesticides, pharmacological agents, and therapeuticagents.

For example, biologically active agents suitable for use in the presentinvention include, but are not limited to, proteins, polypeptides;peptides; hormones; polysaccharides, and particularlymuco-polysaccharides and mixtures thereof; carbohydrates; lipids; otherorganic compounds; and particularly compounds which by themselves do notpass (or which pass only a fraction of the administered dose) throughthe gastro-intestinal mucosa and/or are susceptible to chemical and/orenzymatic cleavage by acids and enzymes in the gastro-intestinal tract;or any combination thereof.

Further examples include, but are not limited to, the following,including synthetic, natural or recombinant sources thereof: growthhormones, including human growth hormones (hGH), recombinant humangrowth hormone (rhGH), bovine growth hormones, and porcine growthhormones; growth hormone-releasing hormones; interferons, including α, βand γ; interleukin-1; interleukin-2; insulin, including porcine, bovine,human and human recombinant, optionally having counter ions includingsodium, zinc, calcium and ammonium; insulin-like growth factor,including IGF-1; heparin, including unfractionated heparin, heparinoids,dermatans, chondroitins, low molecular weight heparin, very lowmolecular weight heparin and ultra low molecular weight heparin;calcitonin, including salmon, eel and human; erythropoietin; atrialnaturetic factor; antigens; monoclonal antibodies; somatostatin;protease inhibitors; adrenocorticotropin, gonadotropin releasinghormone; oyytocin; leutinizing-hormone-releasing-hormone; folliclestimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim;prostaglandins; cyclosporin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferroxamine (DFO); parathyroidhormone (PTH), including its fragments; antimicrobials, includinganti-fungal agents; vitamins; analogs, fragments, mimetics orpolyethylene glycol (PEG)-modified derivatives of these compounds; orany combination thereof.

Modified Amino Acids

The modified amino acid may be an N-acylated or sulfonated amino acid, aketone or aldehyde of an acylated or sulfonated amino acid, saltsthereof, and a polyamino acid or polypeptide which includes any of theforegoing.

N-acylated and sulfonated amino acids, poly amino acids, and peptidesinclude, but are not limited to, amino acids which have been N-acylatedor sulfonated, and poly amino acids and peptides in which at least oneamino acid has been modified, by acylating or sulfonating at least onefree amine group with an acylating or sulfonating agent which reactswith at least one of the free amine groups present.

Preferably, the modified amino acids comprise one of the followingstructures:

and salts thereof, including but not limited to sodium salts.

The modified amino acids may be in the form of salts. Salts include butare not limited to organic and inorganic salts, for example alkali-metalsalts, such as sodium, potassium and lithium; alkaline-earth metalsalts, such as magnesium, calcium or barium; ammonium salts; basic aminoacids such as lysine or arginine; and organic amines, such asdimethylamine or pyridine. Preferably, the salts are sodium salts.

An amino acid is any carboxylic acid having at least one free aminegroup and includes naturally occurring and synthetic amino acids. Polyamino acids are either peptides (which are two or more amino acidsjoined by a peptide bond) or are two or more amino acids linked by abond formed by other groups which can be linked, e.g. an ester,anhydride, or an anhydride linkage. Peptides can vary in length fromdipeptides with two amino acids to poly peptides with several hundredamino acids. One or more of the amino acid or peptide units may beacylated or sulfonated.

N-acylated or sulfonated amino acids are typically prepared by modifyingthe amino acid or an ester thereof. Many of these compounds are preparedby acylation or sulfonation with agents having the formulaX—Y—Rwherein:

R is the appropriate radical to yield the modification indicated in thefinal product,

Y is C═O or SO₂, and

X is a leaving group.

Typical leaving groups include, but are not limited to, halogens suchas, for example, chlorine, bromine, and iodine. Additionally, thecorresponding anhydrides are modifying agents.

N-acylated or sulfonated amino acids can be readily prepared from aminoacids by methods within the skill of those in the art based upon thepresent disclosure. For example, N-acylated or sulfonated amino acidsmay be derived from aminobutyric acid, aminocaproic acid, andaminocaprylic acid. Further, the N-acylated or sulfonated amino acidabove may be prepared by reacting the single amino acid with theappropriate modifying agent which reacts with a free amino moietypresent in the amino acids to form amides. Protecting groups may be usedto avoid unwanted side reactions as would be known to those skilled inthe art.

The amino acid can be dissolved in aqueous alkaline solution of a metalhydroxide, e.g., sodium or potassium hydroxide, and heated at atemperature ranging between about 5° C. and about 70° C., preferablybetween about 10° C. and about 40° C., for a period ranging betweenabout 2 hour and about 4 hours, preferably about 2.5 hours. The amountof alkali employed per equivalent of SHE groups in the amino acidgenerally ranges between about 1.25 and about 3 mmole, preferablybetween about 1.5 and about 2.25 mmole per equivalent of NH₂. The pH ofthe solution generally ranges between about 8 and about 13, preferablyranging between about 10 and about 12.

Thereafter, the appropriate amino modifying agent is added to the aminoacid solution while stirring. The temperature of the mixture ismaintained at a temperature generally ranging between about 5° C. andabout 70° C., preferably between about 10° C. and about 40° C., for aperiod ranging between about 1 and about 4 hours. The amount of aminomodifying agent employed in relation to the quantity of amino acid isbased on the moles of total free NH₂ in the amino acid. In general, theamino modifying agent is employed in an amount ranging between about 0.5and about 2.5 mole equivalents, preferably between about 0.75 and about1.25 equivalents, per molar equivalent of total NH₂ group in the aminoacid.

The reaction is quenched by adjusting the pH of the mixture with asuitable acid, e.g., concentrated hydrochloric acid, until the pHreaches between about 2 and about 3. The mixture separates on standingat room temperature to form a transparent upper layer and a white oroff-white precipitate. The upper layer is discarded, and the N-acylatedor sulfonated amino acid is collected from the lower layer by filtrationor decantation. The crude N-acylated or sulfonated amino acid is thendissolved in water at a pH ranging between about 9 and about 13,preferably between about 11 and about 13. Insoluble materials areremoved by filtration and the filtrate is dried in vacuo. The yield ofN-acylated or sulfonated amino acid generally ranges between about 30and about 60%, and usually about 45%.

If desired, amino acid esters, such as, for example benzyl, methyl, orethyl esters of amino acid compounds, may be used to prepare theN-acylated or sulfonated amino acids of the invention. The amino acidester, dissolved in a suitable organic solvent such asdimethylformamide, pyridine, or tetrahydrofuran is reacted with theappropriate amino modifying agent at a temperature ranging between about5° C. and about 70° C., preferably about 25° C., for a period rangingbetween about 7 and about 24 hours. The amount of amino modifying agentused relative to the amino acid ester is the same as described above foramino acids. This reaction may be carried out with or without a basesuch as, for example, triethylamine or diisopropylethylamine.

Thereafter, the reaction solvent is removed under negative pressure andthe ester functionality is removed by hydrolyzing the N-acylated orsulfonated amino acid ester with a suitable alkaline solution, e.g. 1Nsodium hydroxide, at a temperature ranging between about 50° C. andabout 80° C., preferably about 70° C., for a period of time sufficientto hydrolyze off the ester group and form the N-acylated or sulfonatedamino acid having a free carboxyl group. The hydrolysis mixture is thencooled to room temperature and acidified, e.g. aqueous 25% hydrochloricacid solution, to a pH ranging between about 2 and about 2.5. TheN-acylated or sulfonated amino acid precipitates out of solution and isrecovered by conventional means such as filtration or decantation.Benzyl esters may be removed by hydrogenation in an organic solventusing a transition metal catalyst.

The N-acylated or sulfonated amino acid may be purified byrecrystallization or by fractionation on solid column supports.

Suitable recrystallization solvent systems include acetonitrile,methanol and tetrahydrofuran. Fractionation may be performed on suitablesolid column supports such as alumina, using methanol/n-propanolmixtures as the mobile phase; reverse phase column supports usingtrifluoroacetic acid/acetonitrile mixtures as the mobile phase; and ionexchange chromatography using water as the mobile phase. When anionexchange chromatography is performed, preferably a subsequent 0-500 mmsodium chloride gradient is employed.

Polymers

The polymers of the present invention may be natural or synthetic andcomprise two or more monomers. The monomers may be the same ordifferent, and the polymer may be linear or non-linear. Polymers includebut are not limited to branched or cyclic polymers. The polymers may becopolymers including two or more different monomers, or homo-polymersincluding a single-type of monomeric repeat. Further, polymers may berandom or alternating, directed, bifunctional, polyfunctional,cross-linked, regular lattice, intermittent lattice, or amorphous.

The polymer may be any polymer including, but not limited to,alternating copolymers, block copolymers and random copolymers, whichare safe for use in mammals. Preferred polymers include, but are notlimited to, polyethylene; polyacrylates; polymethacrylates;poly(oxyethylene); poly(propylene); polypropylene glycol; polyethyleneglycol (PEG) and derivatives thereof, such as PEG-maleic anhydridecopolymers; and derivatives and combinations thereof. The molecularweight of the polymer typically ranges from about 100 to about 200,000daltons. The molecular weight of the polymer preferably ranges fromabout 200 to about 10,000 daltons in one embodiment, the molecularweight of the polymer ranges from about 200 to about 600 daltons andmore preferably ranges from about 300 to about 550 daltons.

Polymers may be in the form of one or more salts. Salts include but arenot limited to organic and inorganic salts, for example alkali-metalsalts, such as sodium, potassium and lithium; alkaline-earth metalsalts, such as magnesium, calcium or barium; ammonium salts; basic aminoacids such as lysine or arginine; and organic amines, such asdimethylamine or pyridine. Preferably, the salts are sodium salts

Polymeric Delivery Agent (Conjugates)

One or more of the modified amino acids may be conjugated (covalentlyattached) to one or more of the monomeric units of the polymer via oneof the aforementioned linkage groups.

Many of the polymeric delivery agents have solubility greater than about200 mg/mL, and have greater solubility than the corresponding modifiedamino acids alone. However, like most poloxamers, the solubility of PEGconjugates decreases at higher temperatures and can be characterized bythe cloud point or lower critical solution temperature (LCST). The LCSTis dependent on the ratio of hydrophilic/hydrophobic units in theconjugate and can be changed easily.

In general, the polymeric delivery agents of the present invention maybe prepared as follows. For vinyl polymeric delivery agents, such as PAAand PAA/MA polymers, the polymer and modified amino acids may each beseparately dissolved in an appropriate solvent, e.g., dimethyl formamide(DMF), to yield solutions A and B, respectively. Solution B is warmed toabout 60-70° C., in the presence of a base, e.g. triethylamine. SolutionB is then added to solution A and the mixture is stirred at roomtemperature for 24 hours. The polymeric delivery agents precipitateswith the addition of dilute acid or base and is collected bycentrifugation. The polymeric delivery agents is then hydrolyzed,dialyzed against water, and lyophilized.

The resultant polymeric delivery agents may be analyzed by SizeExclusion Chromatography (SEC) in order to determine the approximatemolecular weight of the polymer and the nitrogen content of theconjugate may be used to approximate the amount of modified amino acidbound to the polymer in the polymeric delivery agent. Preferably, thereis between about 5 and 15% w/w bound modified amino acid in thepolymeric delivery agent, and more preferably, there is between about 10and 15% w/w bound modified amino acid unit in the polymeric deliveryagent.

For PEG delivery agents with ester linkages, a carboxyl-containingdelivery agent reacts with PEG or PEG methyl ether in toluene at150-160° C. in the presence of p-toluene sulfonic acid as a catalyst for3-4 hours. Water generated by the reaction is removed with a Dean-Starktrap. Reverse phase HPLC is used to monitor the reaction. The reactionmixture is washed with saturated NaHCO₃ water solution to removeunreacted starting materials and the catalyst. The polymeric deliveryagents are obtained after evaporation of toluene. The structure isfurther confirmed by nitrogen analysis and ¹H NMR.

PEG delivery agents with amide, amino or urethane linkages may beprepared by reaction of an amino-containing modified amino acids with anappropriately activated polyethylene glycol in pyridine at 70-80° C. for4-5 hours and at room temperature for about 24 hours. Pyridine is thenremoved by evaporation under reduced pressure. The residue is thendissolved in an organic solvent, e.g., methylene chloride, and thesolution is washed with dilute HCl aq., NaCl aq., and NaHCO₃ aq.respectively to remove impurities. Reverse phase HPLC is used to monitorboth the reaction and the work-up process. The polymeric delivery agentsis obtained after evaporation of the organic solvent. The structure isfurther confirmed by nitrogen analysis and ¹H NMR.

In order to prepare PEG delivery agents with urea linkages, a two stepprocess was used. First, a urethane derivative based on the reaction ofan amino terminated hydrophobic compound and 4-nitrophenyl chloroformateis prepared. The reaction is very fast and carried out at roomtemperature in pyridine solution. The intermediate urethane derivativecontains a good leaving group chat can be eliminated on attack ofnucleophilic agents. When this intermediate is reacted with anamino-terminated PEG, both 4-nitrophenol and the PEG adduct with urealinkage were formed.

Delivery Systems

The compositions of the present invention may include one or more activeagents. In one embodiment, the polymeric delivery agents or deliveryagent compounds of the present invention (collectively “deliveryagents”) may be used directly as a delivery agent by simply mixing oneor more delivery agent with the active agent prior to administration.The administration mixtures may be prepared by mixing an aqueoussolution of the delivery agent with an aqueous solution of the activeingredient, just prior to administration. Alternatively, the deliveryagent and the active agent can be admixed during the manufacturingprocess. The solutions may optionally contain additives such asphosphate buffer salts, citric acid, acetic acid, gelatin, and gumacacia.

Stabilizing additives may be incorporated into the delivery agentsolution. With some active agents, the presence of such additivespromotes the stability and dispersibility of the agent in solution. Thestabilizing additives may be employed at a concentration ranging betweenabout 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable, butnon-limiting, examples of stabilizing additives include gum acacia,gelatin, methyl cellulose, polyethylene glycol, carboxylic acids andsalts thereof, and polylysine. The preferred stabilizing additives aregum acacia, gelatin and methyl cellulose.

The amount of active agent is an amount effective to accomplish thepurpose of the particular active agent. The amount in the compositiontypically is a pharmacologically or biologically effective amount.However, the amount can be less than a pharmacologically or biologicallyeffective amount when the composition is used in a dosage unit form,such as a solid, such as a capsule, a tablet, or a powder, or a liquid,because the dosage unit form may contain a multiplicity of deliveryagent or active agent compositions or may contain a dividedpharmacologically or biologically effective amount. The total effectiveamounts can then be administered in cumulative units containing, intotal, pharmacologically or biologically or chemically active amounts ofbiologically or pharmacologically active agent.

The total amount of active agent to be used can be determined by thoseskilled in the art. However, because the presently disclosed deliveryagents provide efficient delivery, lower amounts of biologically orchemically active agent than chose used in prior dosage unit forms ordelivery systems may be administered to the subject, while stillachieving the same blood levels and therapeutic effects.

The amount of delivery agents in the present composition is a deliveryeffective amount and can be determined for any particular deliver,agents or active agent by methods known to those skilled in the art. Itwill be this amount effective for delivery of the active agent by thechosen route of delivery.

Dosage unit forms can also include any of excipients; diluents;disintegrants; lubricants; plasticizers; colorants; and dosing vehicles,including, but not limited to water, 1,2-propane diol, ethanol, oliveoil, or any combination thereof.

Administration of the present compositions or dosage unit formspreferably is oral, intracolonic or intraduodenal. Particularly, thecompositions of the present invention are useful in orally administeringactive agents, especially those that are not ordinarily orallydeliverable.

The delivery compositions of the present invention may also include oneor more enzyme inhibitors. Such enzyme inhibitors include, but are notlimited to, compounds such as actinonin or epiactinonin and derivativesthereof. Other enzyme inhibitors include, but are not limited to,aprotinin (Trasylol) and Bowman-Birk inhibitor.

The compositions of the subject invention are useful for administeringbiologically or chemically active agents to animals. The system isparticularly advantageous for delivering biologically or chemicallyactive agents which would otherwise be destroyed or rendered lesseffective by conditions encountered before the active agent has reachedits target zone (i.e. the area in which the active agent of the deliverycomposition are to be released) and within the body of the animal towhich they are administered.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. Allparts are given by weight unless otherwise indicated. The number ofpolymeric units and molecular weights of the polymers specified in theexamples are an average number of units and average molecular weights.The number of units and molecular weights in the polymers typicallyvaries by up to about lot.

Example 1a Preparation of8-N(2-hydroxy-5-aminomethylbenzoyl)aminocaprylic acid((I-COOH)-5-CH₂NH₂), hydrogen chloride salt

8-N(2-methoxybenzoyl)aminocaprylic acid (7.5 g, 25.6 mmol, 1 equiv.) wasmixed with formaldehyde (30.7 ml, 410 mmol, 16 equiv.) and hydrochloricacid (62.6 ml, 37%, 644 mmol, 25 equiv.). The mixture was stirred andbubbled under HCl gas at room temperature for 3 hours. Crude8-N(2-methoxy-5-chloromethylbenzoyl)aminocaprylic acid (compound 1) wasobtained by removal of formaldehyde and HCl. Pure compound 1 wasobtained by recrystallization in acetonitrile (4.5 g, 51.4%).

The purified 8-N(2-methoxy-5-chloromethylbenzoyl)amino caprylic acid(4.5 g, 13.2 mmol, 1 equiv.) and hexamethylenetetramine (1.85 g, 13.2mmol, 1 equiv.) were refluxed in chloroform for 1 hour. The chloroformwas evaporated. The residue was refluxed in a mixed solution of methylalcohol (30 ml) and HCl (10 ml, 37%) for 2 hours. 8-N(2-methoxy5-aminomethylbenzoyl)aminocaprylic acid (compound 2) was obtained byremoval of the mixed solvent (3.6 g, 76.6%). Compound 2 (3.5 g, 9.75mmol, 1 equiv.) was dissolved in dichloromethane (50 ml). Borontribromide (1.84 ml, 19.5 mmol, 2 equiv.) was added to the reactionmixture at 0° C. and stirred for 2 hours. The product was filtered andthe residue was washed with dichloromethane (20 ml×2). 8-N(2-Hydroxy5-aminomethylbenzoyl)aminocaprylic acid, hydrochloride salt was obtainedas white solid (1.8 g, 47.37%). Properties are listed below:

H NMR (300 Mhz, DMSO-d⁶) δ: 1.29 (6H, br s). 1.5 (4H, m). 2.18 (2H, t, J7.3 Hz). 3.30 (2H, q, J 6.17 Hz). 3.94 (2H, q, J 6.56 Hz). 6.97 (1H, d,J 8.7 Hz). 7.49 (1H, d, J 6.3 Hz). 8.0 (1H, s). 8.12 (3H, br s). 12.0(1H, s). 12.5 (1H, s). Anal. for C₁₆H₂₅N₂O₄Cl: Calculated: C: 55.73; H:7.26; N: 8.12; Cl: 10.30. Found: C: 55.59; H: 7.38; N: 8.01; Cl: 10.18.

Example 1b Synthesis ofN-(5-Aminomethylsalicyloyl)-4-(4-aminophenyl)butyric acid((II-COOH)-5-CH₂NH₂)

Salicylic acid (50 g) was suspended in 120 g of Formalin solution (37%).Hydrogen chloride gas was bubbled through the mixture at 0° C. withmechanical stirring. ZnCl₂ (10 g) was added as the catalyst 5 minuteslater. The hydrogen chloride gas was slowly bubbled through the mixturefor 2 hours (“h” or “hr”) at 0-15° C. and then for another 3 h at roomtemperature with stirring. The reaction mixture was refrigeratedovernight. The precipitate formed was collected by filtration and driedin air. The crude product (75 g, m.p. 115 130° C.) was recrystallizedfrom benzene to give pure product 5-chloromethylsalicylic acid (28-5 g,42%), m.p. 144-147° C.

To a solution of acetic anhydride (1.4 g) and glacial acetic acid (1.7g), 5 chloromethylsalicylic acid (1.9 g) and one drop (using a pipet) ofconcentrated sulfuric acid was added with stirring. The reaction mixturewas heated slowly to 65-70° C. and held for 1 h. After cooling to roomtemperature, the reaction mixture was added gradually to 50 ml of icewater-Two hours later, the precipitate formed was collected byfiltration and dried in vacuo. Recrystallization from benzene gave theproduct O-acetyl-5-chloromethylsalicylic acid (1.7 g, 75%), m.p.119-121° C.

O-Acetyl-5-chloromethylsalicylic acid (2.9 g, 12.7 mmol) and thionylchloride (15 g, 126 mmol) were added to 30 ml of benzene. The mixturewas refluxed for 2 h with stirring. Evaporation of the benzene withexcess thionyl chloride gave a syrupy residue to which 30 ml of benzenewas added and the solvent evaporated again. The residue was dried invacuo overnight to remove residual SOCl₂ from productO-Acetyl-5-chloromethylsalicyloyl chloride.

4-(4-Aminophenyl)butyric acid (5 g) was dissolved in 40 ml methanol. Thesolution was refluxed at 80-90° C. with stirring for 4 h while hydrogenchloride gas was bubbled through the solution. After the reactionmixture was cooled to room temperature, ethyl ether (100 ml.) was added.The mixture, which separated into two layers, was refrigeratedovernight. The crystalline product was collected by filtration and driedthoroughly. The filtrate was evaporated to dryness and the residue wasrecrystallized from MeOH/benzene. The total amount of the product methyl4-(4-aminophenyl)butyrate hydrogen chloride obtained was 5.6 g (87.5%),m.p. 143-145° C.

Methyl 4-(4-aminophenyl)butyrate hydrogen chloride (2.6 g, 11.3 mmol)and triethylamine (2.3 g, 22.6 mmol) were added to 20 ml of methylenechloride (Solution A). O-Acetyl-5-chloromethylsalicyloyl chloride (2.8g, 11.3 mmol) was dissolved in 20 ml. of methylene chloride (SolutionB). The solution A was added dropwise to solution B at 0° C. withstirring. The mixture was stirred at room temperature for another 2 hafter the addition. The reaction mixture was then washed with 0.1 N HClaqueous solution twice (50 ml×2) and saturated NaCl aqueous solutiontwice (50 ml×2). The organic layer was separated and dried overanhydrous sodium sulfate. The solvent was evaporated under reducedpressure to give a syrupy product[N—O-acetyl-5-chloromethylsalicyloyl)]-4-4(4-aminophenyl)butyric acidmethyl ester which was used in the next step without furtherpurification.

The syrupy product obtained above was dissolved in 20 ml of chloroform(Solution C). Hexamethylenetetramine (1.58 g, 11.3 mmol) was dissolvedin 20 ml of warm (about 30° C.) chloroform (Solution D). Solution D wasadded to Solution C and the reaction mixture was refluxed for 2 h at60-80° C. with stirring. The reaction mixture was then allowed to standat room temperature overnight. Evaporation of the solvent gave a syrupof the complex of the previous product with hexamethylenetetramine,which solidified after drying in vacuo for several hours.

The solid complex obtained above was dissolved in 5 ml of MeOH. To thissolution, 5 ml of concentrated HCl solution was added. The reactionmixture was stirred at 40-50° C. for 4 h. The reaction mixture was thenrefrigerated overnight. The precipitate (NH₄Cl) was filtered off. To thefiltrate, 50 ml of MeOH was added. The solvents were evaporated underreduced pressure to give a syrupy product[N—(O-acetyl-5-aminomethylsalicyloyl)]-4-(4-aminophenyl)butyric acidmethyl ester hydrogen chloride which was used in the next deprotectionstep directly without further purification.

The above syrup was dissolved in 5 ml of MeOH. To this solution, 15 mlof 2N NaOH was added. The milk-like solution was stirred at roomtemperature for 4 h, while the pH was kept at 10 to 12 by adding NaOHsolution. The clear solution was acidified to pH 5 to precipitate theproduct, which was then collected by filtration, washed with water andethanol and dried in air. The crude product (3.0 g) was obtained withabout 80% purity checked by ¹H NMR. The crude product was refluxed in 30ml of 95% alcohol for 10 min. and then filtered.

The insoluble substance was dissolved in 20 ml of water at pH 10 to 11.The solution was then acidified to pH 5. The precipitated product wascollected by filtration and dried thoroughly; yield 2.4 g[N-(5-aminomethylsalicyloyl)]-4-(4-aminophenyl) butyric acid((II-COOH)-5-CH₂NH₂) (58% for the last five steps). The m.p. was higherthan 240° C. ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 1.75 (2H,t), 2.2 (2H,t),2.55 (2H,t), 3.95 (2H,s), 7.05 (1H,s), 7.15 (1H,s), 7.5 (1H,d), 7.65(1H,d), 8.18 (1H,s), 8.35 (1H, br.s).

Example 2 Synthesis of I-COOH to XI-COOH

Compound I-COOH may be prepared as follows:

A 3 L three-neck round bottom flask was fitted with an overheadmechanical stirrer and a thermometer, and the flask was cooled in anice-bath. A solution of 8-aminocaprylic acid (100.0 g, 0.65 moles) in 2M aqueous sodium hydroxide (1.4 L) was charged into the round bottomflask. The temperature of the solution was kept at about 5° C. andO-acetylsalicyloyl chloride (198.6 g, 0.76 moles, 1.2 equiv.) was addedportionwise over 7 hours. The mixture was stirred at 5° C. for 12 hoursto yield a yellow homogenous solution. The solution was acidified with 1M hydrochloric acid to pH 6.8 and was extracted with ethyl acetate(2×600 mL). The pH of the aqueous layer was readjusted to 6.3 and wasfurther extracted with ethyl acetate (2×600 mL). The organic layers werecombined, dried over anhydrous sodium sulfate, filtered, and evaporatedunder reduced pressure. The residue was redissolved in a minimum volumeof 2 M aqueous sodium hydroxide, and the pH of the solution was between9.5 and 10. The mixture was acidified with stirring with 1 Mhydrochloric acid to pH of about 6.2, and a solid was formed. The solidwas filtered, washed with water (3×300 mL), and recrystallized from 55%methanol/water (v/v) to yield Compound I-COOH as an off-white solid(99.7 g, 57%). Mp 116-117° C. ¹H NMR (300 MHz, DMSO-d₆), δ: 12.70 (1H,br s), 11.95 (1H, br s) 8.81 (1H, t), 7.82 (1H, m), 7.38 (1H, m), 5.84(2H, m), 2.36 (2H, q), 2.18 (2H, t), 1.50 (4H, br m), 1.28 (6H, m),Anal. Calcd for C₁₅H₂₁NO₄: C, 64.50; H, 7.58; 1 N, 5.02. Found: C,64.26; H, 7.81; N, 4.93.

Compound III-COOH may also be prepared by this same method using10-amino-caprylic acid available from Tyger Scientific, Inc. (MonmouthJunction, N.J.).

Compounds VIII-COOH and IX-COOH may also be prepared by the same methodfor preparing compound I-COOH using the appropriate starting materials.

Compound II-COOH may be prepared as follows. Acetylsalicyloyl chloride(47.00 g, 0.24 mol, 1 equiv.) was added portionwise to a mixture of4-(4-aminophenyl)butyric acid (50.00 g, 0.28 mol, 1.2 equiv.) in aqueoussodium hydroxide (2M, 300 mL). The reaction was stirred at 25° C. for 2hours, and the resultant solution was acidified with aqueoushydrochloric acid (1M to pH 2.1. The resultant precipitate was filtered,and was washed with aqueous hydrochloric acid (1M, 3×100 mL) and waterto give Compound II-COOH as a pale pink solid (31.89 g, 52%). ¹H NMR(300 MHz, DMSO-d₆) δ: 7.74 (1H, dd), 7.38 (2H, d), 7.21 (3H, m), 6.67(1H, m), 6.57 (1H, m), 2.48 (2H, t), 2.07 (2H, t), 1.71 (2H, m). Anal.Calcd for C₁₇H₁₇NO₄: C, 68.20H, 5.73; N, 4.70. Found: C, 68.22; H, 5.61;N, 4.66.

Compounds VI-COOH and VII-COOH may be prepared by this method using theappropriate starting materials.

Compound IV-COOH may be prepared as follows. A slurry of 8-aminocaprylicacid (75.0 g, 0.471 mmol) in methylene chloride (500 mL) was treatedwith chlorotrimethylsilane (102.34 g, 0.942 mol) and was heated toreflux for 2 hours. The reaction mixture was cooled to 0° C. and wasthen treated with triethylamine (142.98 g, 1.413 mol) followed by thedropwise addition of 4-methoxy-2-acetylbenzoyl chloride (107.71 g, 0.471mol). The reaction mixture was stirred for 0.5 hours at 0° C. and thenfor 2 days at 25° C. Methylene chloride was removed in vacuo. NaOHsolution (2N) was added to the residue. This mixture was allowed to stirfor 2 hr before the mixture was acidified to pH=1 with concentratedhydrochloric acid. The resulting solid was recovered by filtration andrecrystallized in methylene chloride/hexane (1/1) several times. Thestructure was confirmed by ¹H NMR. 38% Yield cleanproduct-8-N-(4-methoxysalicyloyl)aminocaprylic acid (59.58 g, 0.193mol).Compound V-COOH May be Prepared as Follows.

1,8-Diaminooctane (1.44 g, 10 mmol) was dissolved in 50 ml oftetrahydrofuran (THF). To the solution was added dropwise succinicanhydride (1.0 g, 10 mmol) in 20 ml of THF at room temperature withstirring. A precipitate formed immediately. The reaction mixture wasstirred for another 30 min. after addition. The precipitate wascollected by filtration, washed thoroughly with THF and dried in air. Itwas dissolved in 10 ml of water at pH 10. The pH was then adjusted to 1with 1N HCl water solution. The precipitate was filtered off. Thefiltrate was lyophilized to give a solid powder, which was thenextracted with ethanol. Evaporation of ethanol gave 8-aminooctylsuccinicmonoamide hydrogen chloride 2.2 g (78%), which was used for nextreaction without further purification. 8-Aminooctylsuccinic monoamidehydrogen chloride (2.2 g, 7.8 mmol) was dissolved in 25 ml of 1N NaOHwater solution. To the solution was added O-acetylsalicyloyl chloride(1.55 g, 7.8 mmol) in three portions over a 2 hour period at roomtemperature with stirring. The mixture was stirred for another 2 hours.The pH of the reaction mixture was adjusted to 7. The precipitate formedwas filtered off. The pH of the filtrate was then adjusted to 2. Thesolution was kept at room temperature for 2 hours. The precipitate wascollected by filtration and dried in air. It was purified byrecrystallization from ethanol/water, yield 1.5 g (55%)8-Salicyloylaminooctyl succinic monoamide compound V-COOH, m.p. 123-125°C. Its structure was confirmed by reverse phase HPLC (t_(R)=4.3 min.),elemental analysis and ¹H NMR. Elemental Analysis for C₂₉H₂₈N₂O₅:Calculated: C, 62.63; H, 7.69; N, 7.69. Found: C, 62.84; H, 7.60; N,7.60. ¹H NMR (300 MHz, DMSO-d₆, ppm): 12.7 (br, 1H), 12.0 (br, 1H), 8.8(t, 1H), 7.8 (q, 1H), 7.35 (h, 1H), 6.8 (q, 2H), 3.2 (m, 2H), 2.95 (m,2H), 2.35 (t, 2H), 2.2 (t, 2H), 1.5 (m, 2H) and 1.2 (m, 10H).

Compounds X-COOH and XI-COOH are commercially available from Aldrich(Milwaukee, Wis.).

Example 3 Preparation of Conjugate 1

Acryloyl chloride (45 ml) was added dropwise to a solution ofN-hydroxysuccinimide (57-6 g) in 77 ml of triethylamine and 750 ml ofchloroform at 0° C. over 40 minutes. The reaction mixture was stirredfor an additional 40 minutes at room temperature, and then was washedwith 300 ml of ice water and 300 ml of saturated NaCl solution. Theorganic layer was dried over anhydrous sodium sulfate in the presence of50 mg of 4-butylpyrocatechol. After filtration, the filtrate wasevaporated to 100 ml, to which 350 ml of n-hexane/ethyl acetate (6:1)was added with vigorous stirring to precipitate the product. The mixturewas refrigerated overnight, and the precipitate was filtered and driedin vacuo. Recrystallization from ethyl acetate/hexane (1:1) gave 55 g ofpure product N-acryloxysuccinimide, m.p. 69-70° C. An additional 12 g ofthe product was isolated from the mother liquor. The total yield was91%.

15 g (0.89 mol) of product N-acryloxysuccinimide and 81 mg AIBN(2,2′-azobisisobutyronitrile) (0.49 mmol) were dissolved in 100 ml ofbenzene. Nitrogen gas was bubbled through the solution for 10 minutesand the flask was sealed. The reaction mixture was placed in an oil bathat 60° C. for 24 hours. The polymer precipitate was filtered, washedwith benzene, and dried in vacuo. The yield ofpoly(N-acryloxysuccinimide) was 15 g (quantitative).

3.75 g (22 mmol) of product poly(N-acryloxysuccinimide) was dissolved in55 ml of DMF (solution E). 0.8 g (2.6 mmol) ofN-(5-aminomethylsalicyloyl)-8-aminocaprylic acid prepared as in Example1 were dissolved at 60-70° C. in 55 ml DMF and 0.52 g of triethylamine(solution F). The warm solution F was added gradually to solution E andthe mixture was stirred at room temperature for 24 hours. The polymerconjugate of poly(N-acryloxysuccinimide) andN-(5-aminomethylsalicyloyl)-8-aminocaprylic acid (“unhydrolyzed polymerconjugate”) was precipitated with dilute 0.1N HCl and collected bycentrifugation. Then, unhydrolyzed polymer conjugate was hydrolyzed in40 ml of 4% NaHCO₃ solution for 48 hours at room temperature anddialyzed against water for 48 hours using Spectra/Por dialysis membrane(MW cut-off 1000)(Spectrum Inc., Laguna Hills, Calif.). The solution waslyophilized and dried to give 2.23 g (85%) of conjugate 1(I-COOH)-5-CH₂NH—PAA. No low molecular weight compounds were found withSEC analysis. Mw=225,300, Mn=131,300, Mw/Mn=1.72. The nitrogen contentin the conjugate was 1-39%, which corresponds to 14.7% of the deliveryagent in the conjugate.

Example 4 Preparation of Conjugate 2

(II-COOH)-5-CH₂NH-PAA conjugate was prepared by the method outlined inExample 3, except thatN-(5-aminomethylsalicyloyl)-4-(4-aminophenyl)butyric acid from Example 2was used in place of the product from Example 1, and after conjugationthe polymer was precipitated with 2% NaHCO₃ solution (rather than 0.1NHCl). The yield was 80%. SEC analysis: Mw=210,900, Mn=118,600,Mw/Mn=1.79. The nitrogen content of the conjugate was 0.951 or 12% ofthe delivery agent in the conjugate.

Example 5 Preparation of Conjugate 3

Alternating cc-polymer of M-PEG allyl ether and maleic anhydrideavailable from Shearwater Polymers, Inc. (Huntsville, Ala.) was used.The co-polymer has an average molecular weight of PEG chain in theco-polymer unit of about 1500 D, and a total molecular weight of 14,000D-2.45 g of the co-polymer was dissolved in 30 ml of DMF. 464 mg of theproduct from Example 1 (I-COOH-5-CH₂NH₂) was dissolved in 70 ml of DMFwhere 2 ml Et₃N have been added, and both solutions were mixed togetherand stirred at room temperature for 24 hours. The reaction was monitoredby HPLC analysis. Then the solution was reduced in vacuo. The solid wasdissolved in 100 ml of water and the pH of the solution was adjusted to10.5-11.0. This solution was dialysed against water (Mw_(cutoff)=3,000)for a total of 72 hours. 2.05 g (70%) of (I-COOH)-5-CH₂NH-PEG/maleicanhydride was obtained. The final product was analyzed by reverse phaseHPLC, SEC and NMR. Based on HPLC data, it contained no more than 1.5% ofthe product from Example 1. SEC data; M_(w) 20,100 M_(n) 11,200 Mw/Mn1.79. The nitrogen content in the conjugate was 1.07% or 10.5% w/w ofthe delivery agent bound to the polymer.

Example 6 Poly(ethylene glycol)[PEG]-Delivery Agent Conjugates PreparedVia Esterification Reaction

A number of PEG-delivery agent conjugates of different molecular weightand functionality have been prepared using linear mono- and dihydroxyterminated PEG MW=200-4,500 and branched polyfunctional PEG of molecularweight 1,770 and 10,000 available from Shearwater Polymers, Inc.(Huntsville, Ala.). The examples of the structures of PEG and thedelivery agents used for the esterification reaction are indicatedbelow:

PEG linear: HO—(CH₂CH₂—O)_(m)—R m=4-100, R═H, OCH₃

PEG branched (8 arms):

Example 6a

The reaction was carried out in a 0.5 L round bottom flask equipped witha condenser and a Dean-Stark trap. 30 g of PEG MW=300 (Carbowax 300) and5 g of I-COOH were dissolved in 175 mL of toluene. 0.6 g of the catalystp-toluene sulfonic acid monohydrate was added. The solution was refluxedfor 30-40 min. and then a second portion of I-COOH (5 g) was added. Thereaction was monitored by reverse phase HPLC and was stopped when only3-5% of non-reacting I-COOH was found on the chromatogram. This normallytakes 3-4 hours, and in this case took approximately 3 hours. Thesolution was cooled to room temperature and poured into 750 mL ofslightly basic water (pH=7.5-8.0 adjusted with NaHCO₂ saturatedsolution) and was left for 3-4 hours in the separation funnel. Threelayers formed: a top toluene layer, an intermediate water layer and abottom layer containing the target conjugate. The bottom layer wasseparated and was reduced under vacuum. 13.2 g (63%) of oily product wasobtained. NMR confirmed the conjugate's structure: —COOCH ₂— δ=4.13 ppm(tripl.) and the absence of COOH group. The content of nitrogen was2.54% (calculated 2.53%). The calculated molecular weight of theconjugate is −600. The structure of the conjugate is shown below.

I-COO—C₂CH₂O(CH₂CH₂O)₅CH₂CH₂OH Conjugate 4

The following conjugates were prepared by the same method using theappropriate starting materials. The molecular weights of the PEG aregiven in parentheses Conjugates 14 (300), 15 (300), 3.6 (400), 24 (350),25 (550), 26 (350), 27 (400), 28 (300), 29 (400), 32 (2,000), 33 (600),35 (10,000), 36 (10,000), 37 (10,000), 38 (10,000), 39 (2,000) (theproduct precipitated out in a separation funnel), 41 (300)(the productprecipitated out in a separation funnel), 42 (300), 43 (550), 44 (600),45 (1,000), 46 (1,000), 47 (600), were prepared by this method withappropriate starting materials

Example 6b

The same procedure as in 6a was used to prepare a conjugate based onCarbowax200 and the delivery agent I-COOH. The yield of the conjugatewas 67%. Nitrogen content was 3.01%. The calculated molecular weight is470 D. The structure is shown below.

I-COO—CH₂CH₂O(CH₂CH₂O)₃CH₂CH₂OH Conjugate 5

Example 6c

The reaction was carried out using the same equipment as in 6a. 5.6 g ofpoly(ethylene glycol) methyl ether MW-350 (Aldrich) and 3.6 g of I-COOHwere dissolved in 80 mL of toluene. 0.3 g of catalyst p-toluene sulfonicacid monohydrate was added. The solution was refluxed for 3 hours. Thesolution was then cooled to room temperature and transferred into aseparation funnel. A mixture of 53 mL of water and 7 mL of saturatedsolution of NaHCO₃ were added. The top toluene layer was separates,washed with water and reduced under vacuum. The final product was aviscous oil, yield was 92%, and nitrogen content was 2.25%. Thecalculated molecular weight is 620 D. The structure is shown below:

I-COO —CH₂CH₂O(CH₂CH₂O)₅CH₂CH₂OCH₃ Conjugate 6

The following conjugates were prepared by the same method using theappropriate starting materials. The molecular weights of the PEG methylether are given in parentheses conjugates 13 (750), 21 (550), 40 (1,900)and 48 (350).

Example 6d

The same procedure as in 6a was used to prepare a conjugate based onCarbowax400 and the delivery agent I-COOH. The yield of the conjugatewas 63%. Nitrogen content was 2.4%. The calculated molecular weight is700 D. The structure is shown below.

I-COO—CH₂CH₂O(CH₂CH₂O)₇CH₂CH₂OH Conjugate 7

Example 6e

The same procedure as in 6a was used to prepare a conjugate based onCarbowax300 (PEG MW=300) and delivery agent II-COOH. The yield of theconjugate was 65%, nitrogen content-2.06. The calculated molecularweight is 600 D. The structure is shown below:

II-COO—CH₂CH₂(CH₂CH₂O)₅CH₂CH₂OH Conjugate 8

Conjugates 19 and 20 were prepared by this same method except that PEGmethyl ether with MW=550 and MW=350, respectively, was used instead ofPEG MW=300.

Example 6f

The same procedure as in 6a was used to prepare a conjugate based onCarbowax400 and the delivery agent III-COOH. The yield of the conjugatewas 61%. Nitrogen content was 1.98%. The calculated molecular weight is700. The structure is shown below.

III-COO—CH₂CH₂O(CH₂CH₂O)₇CH₂CH₂OH Conjugate 9

Example 6g

The same procedure as in 6a was used to prepare a conjugate based onCarbowax600 and the delivery agent I-COOH. The yield of the conjugatewas 71%. Nitrogen content was 1.05%. The calculated molecular weight is900. The structure is shown below.

I-COO—CH₂CH₂O(CH₂CH₂O)₁₁CH₂CH₂OH Conjugate 10

Example 6h

15.0 g (0.0065 equiv.) of PEG MW=4600 (“Carbowax 4600NF”), 1.65 g(0.0058 equiv.) I-COOH and 0.45 g of p-toluene sulfonic acid monohydratewere dissolved in 120 mL of toluene. The reaction mixture was refluxedfor 13 hours. The reaction was monitored by HPLC and was stopped when anegligible amount of non-reacting I-COOH remained. The solution wasreduced under vacuum. 15.3 g (92%) of solid waxy product was recovered.The conjugate was identified by NMR, HPLC and elemental analysis(N-0.43%). The calculated molecular weight is 5,100. This conjugate hasthe structure below:

I-COO—CH₂CH₂O(CH₂CH₂O)₁₀₂CH₂CH₂—OOC—I Conjugate 11

Example 6i

6.3 g of branched 8 arms PEG2000 (actual MW=1770), 4.0 g I-COOH and 0.2g of p-toluene sulfonic acid monohydrate was refluxed in 30 mL oftoluene for 2.5 hours. 9.9 g of a viscous oily product was obtainedafter toluene evaporation. Based on HPLC data, the conjugate was foundto contain only a negligible amount of non-reacting original I-COOH. NMRspectrum shows the presence of an ester group (δ=4.2 ppm) and confirmsthe absence of carboxyl groups in the conjugate. The yield was 85% andthe Nitrogen content was 0.62%. The calculated molecular weight is 2800.The compound I-COO is attached at the PEG-OH group through an esterlinkage at 4 of the 8 “arms”. The conjugate is denoted “conjugate 12”,

Example 7 Preparation of Compound I-CH₂NH₂ and its Hydrogen ChlorideSalt

O-Acetylsalicylic acid (36 g, 0.2 mol) and N-hydroxysuccinimide (23 g,0.2 mol) were dissolved in 200 ml of DMF. 1,3-Dicyclohexylcarbodiimide(40.5 g, 0.2 mol) in 50 ml of chloroform was added dropwise to thesolution at 0° C. with stirring. The mixture was stirred for another 20h at room temperature. The precipitate was filtered off. Evaporation ofthe filtrate under reduced pressure gave a syrupy residue. It wasre-dissolved i.r. 200 ml of chloroform. The solution was kept in freezerfor 2 h. The precipitate was again filtered off. The filtrate was washedwith 4% sodium bicarbonate (200 ml×3), 10% NaCl (200 ml×2), 0.1N HCl(200 ml×2) and 10% NaCl (200 ml×2) water solutions, respectively. It wasdried over anhydrous sodium sulfate. Evaporation of chloroform gave asyrupy product (50 g, 90%), which solidified after being dried in vacuoovernight.

The active ester obtained above (25 g, 90 mmol) in 150 ml of methylenechloride was added dropwise to a solution of 1,8-diaminooctane (26 g,180 mmol) in 500 ml of methylene chloride over a period of 2 to 3 h (onedrop per second). The mixture was stirred for an additional 15 h. Theprecipitate was collected by filtration, washed with methylene chlorideand dried in air. It was extracted with 300 ml of 0.1N HCL watersolution for 30 min. with stirring. The insoluble substance was filteredoff. The filtrate was adjusted to pH 8 to 9 with 40% sodium hydroxide.Precipitation occurred at this point. It was kept at room temperaturefor 3 h. The precipitate was collected by filtration. It was driedthoroughly in air, yield log(42%), m.p, 155-156° C. This compound can befurther purified by recrystallization from ethanol or water. Reversephase HPLC: t_(R)=2.85 min. ¹H NMR (300 MHz, DMSO-d6), δ(ppm): 1.25 (8H,m), 1.45 (4H, m), 2.1 (2H, t), 3.2 (2H, m), 6.45 (1H, h), 6.6 (1H, q),7.1 (1H, h), 7.7 (1H, q), 6.5 (3H, br), 10.8 (1H, br). Elementalanalysis for C₁₅H₂₄O₂N₂ (+0.25H2O): calculated: C, 67.04; H, 9.12; N,10.43; found: C, 67.13; H, 9.37; N. 10.64. The water content wasdetermined by KF (0.25%).

The product from above Compound I-CH₂NH₂ (0.3 g, 1.1 mmol) was dissolvedin 10 ml of warm (about 50° C.) anhydrous ethanol. Anhydrous hydrogenchloride gas was bubbled through the solution for 10 min. Dry air wasthen passed through the solution for 10 min. Evaporation of the solventgave a solid residue, which was recrystallized from EtOH/Et₂O to givethe hydrogen chloride salt of Compound I-CH₂NH₂ (0.31 g, 91%), m.p.119-121° C. Reverse HPLC: t_(R)=2.9 min.; ¹H NMR (300 MHz, DMSO-d₆),δ(ppm): 1-2 (8H, m), 1.5 (4H, m), 2-7 (2H, m), 3.2 (2H, m), 6.85 (2H,m), 7.35 (1H, h), 7.8 (1H, q), 7.9 (3H, br), 8.9 (1H, t), 12.7 (1H, br).Elemental analysis for C₁₅H₂₅O₂N₂Cl: calculated: C, 59.90; H, 8.32; N,9.32; found C, 60.02; H, 8.21; N. 9.28.

Example 8 Preparation of Conjugate 17

Monomethoxy polyethylene glycol 350 (2 g, 5.7 mmol) was dissolved in 20ml of methylene chloride containing 4 ml of pyridine. To this solutionwere added 1.2 g (5-mmol) of 4-nitrophenylchloroformate and 85 mg of4-dimethylaminopyridine as catalyst at 0° C. The reaction mixture wasstirred for 2 h at 0° C. and another 2 h at room temperature. Thereaction was monitored with reverse phase HPLC. Evaporation of methylenechloride gave the syrupy product, which was used directly for the nextstep reaction without further purification.

1-N-Salicyloyl-1,8-diaminooctane (1.5 g, 5.7 mmol) prepared as above inExample 7 was dissolved in 25 ml of 70-80° C. pyridine. The solution wasmixed with the p-nitrophenyl monomethoxy polyethylene glycol 350carbonate obtained from the previous reaction. The reaction mixture wasstirred at room temperature for 50 h. Evaporation of pyridine underreduced pressure gave a syrupy raw product. It was dissolved in 200 mlof methylene chloride. The solution was washed with 0.1N HCl (200 ml×3),10% NaCl (200 ml×2), 4% sodium bicarbonate (200 ml×3) and 10% NaCl (200ml×2) water solutions. It was dried over anhydrous sodium sulfate.Reverse phase HPLC was used to monitor both the reaction and the work-upprocess. Evaporation of methylene chloride gave the product 1.9 g (53%).The structure was confirmed by reverse phase HPLC (tR=5.43 min.), Nanalysis (the calculated value is 4.32%, the found value was 4.04%) andNMR. A new triplet peak for one amide proton was observed at 7.2 ppm;the chemical shift of methylene proton linked to the free amino group ofthe starting material shifted from 2.6 ppm to 3.0 ppm when the aminogroup was converted to the urethane. The calculated molecular weight is640.

Conjugate 34 was also prepared by this method using the product asprepared in Example 1 and PEG MW=2,000.

Example 9 Preparation of Conjugate 18

Monomethoxy polyethylene glycol 350 (3.5 g, 10 mmol) was dissolved in 20ml of chloroform containing 1.8 g (18 mmol) of triethylamine. To thissolution, 2.0 g of tosyl chloride (10.5 mmol) was added with stirring.The reaction mixture was stirred overnight at room temperature. Theprecipitate was filtered off. The filtrate was diluted with 100 ml ofchloroform. The solution was then washed with 0.1N HCl (100 ml×3), 10%NaCl (100 ml×2), 4% sodium bicarbonate (100 ml×2) and 10% NaCl (100ml×2) water solutions. The chloroform layer was dried over anhydroussodium sulfate. Evaporation of chloroform gave the tosylated monomethoxypolyethylene glycol 350, which was used for the following reaction.

1-N-Salicyloyl-1,8-diaminooctane (2.64 g, 10 mmol), D32, was dissolvedin 30 ml of 70-80° C. pyridine. To this solution, the tosylatedmonomethoxy polyethylene glycol 350 obtained from the previous reactionin 20 ml of pyridine was added dropwise over 40 minutes with stirring.The reaction was stirred at 70° C. for 5 h and at room temperatureovernight. The precipitate was filtered off. The filtrate was evaporatedunder reduced pressure to dryness to give a syrup raw product, which wasdissolved in 150 ml of methylene chloride. The solution was washed with0.1N HCl (150 ml×3) and 10% NaCl (150 ml×2) water solutions. Themethylene chloride solution was collected and dried over anhydroussodium sulfate. After evaporation of methylene chloride, an oilysubstance was obtained. It was then dissolved in 200 ml of water. Themilk solution obtained became clear after extraction with diethyl etherfive times (200 ml×5). The clear water solution was then extracted with200 ml of methylene chloride. The methylene chloride layer was collectedand dried over anhydrous sodium sulfate. Both the reaction and thework-up process were monitored by reverse phase MPLC. Evaporation ofmethylene chloride gave the product 0.8 g (29%). The structure wasconfirmed by reverse phase HPLC (t_(R)=3.73 min.), N analysis (thecalculated value is 3.65%, the found value was 3.39%) and NMR. The peakof two protons of amine salt was observed at 8.5 ppm. Four aromaticprotons of p-toluenesulfonic acid were found at 7.1 ppm and 7.5 ppm. Twomethylene protons of the methylene group linked to the free amino groupof the starting material shifted from 2.6 ppm to 3.6 ppm when the aminogroup was converted to the p-toluenesulfonic acid salt form. Thecalculated molecular weight is 768.

Example 10 Preparation of Conjugate 22

2-(monomethoxy polyethylene glycol 350)acetic acid (MW=350) (12.0 g,29.4 mmol) in 50 ml of methylene chloride was added in a solution of 4.0g (35 mmol) of N-hydroxysuccinimide in 8 ml of DMF and 20 ml ofmethylene chloride. To this solution, 7.4 g (36 mmol) of DCC in 30 ml ofmethylene chloride was added. The reaction mixture was stirred at roomtemperature for 24 h. The precipitate was filtered off. The filtrate waskept in the freezer for 2 h. Again the precipitate was filtered off. Thefiltrate was diluted with 100 ml of methylene chloride The solution waswashed with 0.1N HCl (200 ml×3), 10% NaCl (200 ml×2), 4% sodiumbicarbonate (200 ml×3) and 10% NaCl (200 ml×2) water solutions and driedover anhydrous sodium sulfate. Evaporation of methylene chloride gavethe PEG acetic acid active ester intermediate 8.8 g (58%), which wasused for the following reaction without further purification

1-N-Salicyloy-1,8-diaminooctane (3.6 g, 13.6 mmol) was dissolved in 45ml of 70-80° C. pyridine. To the solution was added 6.9 g (13.6 mmol) ofthe PEG acetic acid active ester prepared above. The reaction mixturewas stirred under nitrogen atmosphere for 4 h at 70° C. It was then keptat room temperature overnight. Reverse phase HPLC was used to monitoredthe reaction. Evaporation of pyridine gave a syrupy raw product. It wasdissolved in 200 ml of methylene chloride. The solution was washed with0.1N HCl (200 ml×3), 10% NaCl (200 ml×2), 4% sodium bicarbonate (200ml×3) and 10% NaCl (200 ml×2) water solutions. The methylene chloridesolution was collected and dried over anhydrous sodium sulfate.Evaporation of methylene chloride gave the syrupy product (6.6 g, 74%),which was further purified as follows. The syrupy product was dissolvedin 200 ml of distilled water. The solution was refrigerated (5-10° C.)overnight. The precipitate was then carefully filtered off until a clearfiltrate was obtained. The solution was extracted with 100 ml ofmethylene chloride twice. The methylene chloride solution was dried overanhydrous sodium sulfate. Evaporation of methylene chloride gave theproduct 6.3 g. Trace amount of methylene chloride was removed from thesyrupy product by bubbling nitrogen through the product. The chemicalstructure was confirmed by reverse phase HPLC (t_(R)=4.6 min.), nitrogenanalysis (the calculated value is 4.28%, the found value was 4.18) andNMR. A new triplet peak for one amide proton was observed at 7.65 ppm.The chemical shift of methylene proton linked to the free amino group ofthe starting material shifted from 2.6 ppm to 3.1 ppm when the aminogroup was converted to the amide. The calculated molecular weight is654.

Conjugate 23 was also prepared by this method except that 2-(monomethoxypolyethylene glycol 550) acetic acid (MW=550) was used instead of theMW=350 material.

Conjugate 30 was also prepared by this method except that the startingmaterials were 2-(monomethoxy polyethylene glycol 350) acetic acid andthe product from Example 1 (I-COOH)-5-CH₂—NH₂)(instead of1-N-salicyloyl-1,8-diaminooctane).

Example 11 Preparation of Conjugate 31

5.0 g (18.9 mmol) of 1-N-salicyloyl-1,8-diaminooctane (CompoundI-CH₂NH₂) prepared as in Example 7 was dissolved in 100 mL of 90-100° C.pyridine, and then cooled to room temperature. The resulting suspensionof N-salicyloyl-1,8-diaminooctane was added by pipet to 5.73 g (28.4mmol) of 4-nitrophenyl chloroformate dissolved in 100 mL of pyridine andstirred for 15 minutes. The solvent was removed by evaporation leaving ared oil with precipitate. The oil was dissolved in 100 mL ofdichloromethane and the precipitate was filtered off. Thedichloromethane solution was washed with 0.1 N HCl (100 ml, threetimes), 5% sodium bicarbonate solution (75 mL, three times), dried oversodium sulfate and evaporated to yield 4.3 g of an orange-brown solid at53% yield ofN-(4-oxycarbonyl-nitrobenzene)-N-Salicyloyl-1,8-diaminooctane.

The above product was then reacted withmethoxypoly(oxyethylene/oxypropylene)-2-propylamine HTJ-506 manufacturedby Huntsman (Houston, Tex.) (hereafter referred to asmethoxyPEG_(1,000)-NH₂). 1.95 g (4.54 mmol) of the above product wasdissolved in 20 mL of acetonitrile and a precipitate (approx. 220 mg)was filtered off. The solution was added to 4.90 g (4.55 mmol)methoxyPEG_(1,000)-NH₂ dissolved in 20 mL of acetonitrile. The reactionwas monitored with HPLC and completed after 3.5 hours at roomtemperature. The solvent was removed by evaporation and 6.4 g of ayellow-orange oil was found. The oil was dissolved in 40 mL ofdichloromethane. This solution was washed twice with 50 mL of 0.1N HCl(to remove unreacted methoxyPEG_(1,000)-NH₂), once with 10% NaClsolution, and continuously washed with 50 mL portions of 5% sodiumbicarbonate solution until 4-nitrophenyl was removed (as detected byreverse phase HPLC). The solution was dried over sodium sulfate andevaporated to yield 3.5 g of a yellow-orange solid This solid was thendissolved in 50 mL of distilled water and washed with 30 mL of diethylether. The water layer was collected and the final product was extractedfrom water with dichloromethane, dried over sodium sulfate andevaporated to yield 3.1 g of a yellow-orange wax at 57% yield ofConjugate 31. It has been named N-PEG-N′-(N-salicyloyl)-heptylamineurea. The purity and structure was confirmed by reverse phase HPLC,elemental analysis (calculated value of N is 3.02%, found 3.09%) andNMR. Two new multiplet peaks at δ=5.57 ppm and 5.79 ppm of equalintensity which are characteristic for substituted ureas were observed,and the peak of methylene proton next to amino group of startingmaterial at 2.6 ppm disappeared. The calculated MW is ˜1400.

Example 12 Recombinant Human Growth Hormone (rhGH) Oral/IntracolonicDelivery

Oral gavage (PO) and/or intracolonic (IC) dosing solutions of deliveryagent compound and rhGH in water, phosphate buffer (PB) or 5% aqueousethanol were prepared. Typically, a solution of the conjugate wasprepared by mixing in water and stirred. The final dosing solutions wereprepared by mixing the conjugate solution with an rhGH stock solution(typically 15 mg rhGH/ml) and diluting to the desired volume (usually3.0 ml). The compounds and rhGH dose amounts are listed below in Table1.

Male Sprague-Dawley rats weighing between 200-250 g were fasted for 24hours and administered ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing. A dosing group of five rats wasadministered one of the dosing solutions. For oral gavage (PO), an 1 mlRusch 8 French catheter was adapted to a 1 ml syringe with a pipettetip. The syringe was filled with dosing solution by drawing the solutionthrough the catheter, which was then wiped dry. The catheter was placeddown the esophagus leaving 1 cm of tubing past the rat's incisors.Solution was administered by pressing the syringe plunger. Forintracolonic (IC) dosing, a 7.5 cm Rusch catheter tube (French 8 or 6)was adapted to a syringe with an Eppendorf pipette tip. The syringe wasfilled with the 0.5 ml dosing solution by drawing the solution throughthe catheter tube. The catheter tube was wiped dry. K-Y jelly wasapplied to the tip, avoiding contact with the eye of the tube, and thetube was inserted into the colon through the anus until the tube was nolonger visible. The solution was injected by pressing the syringeplunger, and the tube was removed.

Blood samples were collected serially from the tail artery, typically attime=0, 15, 30, 45, 60 and 90 minutes for oral and 0, 10, 20, 30, 60 and90 for IC dosing. Serum rHGH concentrations were quantified by an rHGHimmunoassay test kit (Kit # K1F4015 from Genzyme Corporation Inc.,Cambridge, Mass.). Previous studies indicated baseline values of aboutzero.

The results of PO administration are presented in Table 1 below whereinrhGH was administered with (a) delivery agent I alone, (b) Conjugate 1,and (c) Conjugate 3. The experiments were performed at 1/10 the deliveryagent concentration versus that of the conjugate. Thus, at a dose of 200mg/kg conjugate, the actual amount of delivery agent dosed was 20 mg/kg.With such a concentration of delivery agent complexed with polymer therewas evidence of systemic delivery.

TABLE 1 Oral Administration of rhGH Base Rat. Group line Min15 Min30Min45 Min60 Min90  1 7625 (a) 0 84.73 13.19 0 10.565 0 −1  2 −2 (a) 079.98 22.69 0 5.49 0  3 −3 (a) 0 48.72 1.82 0 7.905 0  4 −4 (a) 0 45.3446.52 35.53 34.885 2.715  5 −5 (a) 0 0 58.44 0 3.922 0  6 7626 (a) 064.2 18.99 4.29 10.13 0 −1  7 −2 (a) 0 43.14 23.44 18.35 15.93 1.47  8−3 (b) 0 6.16 0 0 4.92 0  9 −4 (b) 0 30.71 0 0 10.025 0 10 −5 (b) 026.15 0 0 9.985 0 11 7627 (b) 0 5.35 0 0 4.28 0 −1 12 −2 (b) 0 14.99 0 03.39 0 13 −3 (b) 0 13.74 0 0 6.075 0 14 −4 (b) 0 16.05 0 0 21.495 0 15−5 (c) 0 14.42 0.1 0 11.26 0 16 7628 (c) 0 7.87 0 0 15.19 0 −1 17 −2 (c)0 14.31 0 0 17.545 0 18 −3 (c) 0 0 3.57 0 4.745 0 19 −4 (c) 0 28.7683.12 0 2.78 0 20 −5 (c) 0 5.08 0 0 0.235 0

Dose volume for IC administration was 1 ml/kg. rhGH dose was 1 mg/kg.For IC dosing, the five samples from each time period were pooled andthe maximum concentration for each group (Cmax) are reported below inTable 2.

TABLE 2 Intracolonic Delivery of rhGH in Rats Dosing Conju- rhGH MeanPeak Conju- solution gate Dose Dose Serum [rhGH] gate medium (mg/kg)(mg/kg) (ng/ml) ±SD  1 PB 25 1 16 ± 9   1 PB 25 1 1 ± 2  3 PB 25 1 14 ±34  4 PB 25 1  6 ± 16  4 5% aq. 25 1 182 ± 18  EtOH  4 5% aq. 61 1 172 ±29  EtOH  6 5% aq. 25 1 168 ± 54  EtOH  7 5% aq. 25 1 205 ± 95  EtOH  85% aq. 25 1 101 ± 32  EtOH 10 water 120  1 30 ± 23 10 PB 25 1 0.5 ± 1.012 5% aq. 25 1 0 EtOH 13 5% aq. 25 1 63 ± 43 EtOH 14 5% aq. 25 1 75 ± 35EtOH 17 5% aq. 25 1 136 ± 37  EtOH 18 5% aq. 25 1 140 ± 51  EtOH 22 5%aq. 25 1 164 ± 53  EtOH 24 PB 25 1 0 25 PB 25 1 0

Example 13 Parathyroid Hormone Delivery (PTH 1-34)

Oral/Intracolonic Deliver

Oral gavage (PO) and/or intracolonic (IC) dosing solutions of deliveryagent compound and human parathyroid hormone residues 1-34 (PTH) inwater and various aqueous solutions as indicated in the Table 3 below(PEG 300 and PEG 350 is available from Aldrich, (Milwaukee, Wis.).Kollidon 17PF is polyvinyl-pyrrolidone available from Aldrich. PG ispropylene glycol). Typically, a solution of the conjugate was preparedin the appropriate medium and stirred. The final dosing solutions wereprepared by mixing the conjugate solution with a PTH stock solution(typically 5 mg PTH/ml) and diluting to the desired volume (usually 3.0ml). The final compound, PTH and volume dose amounts, and the dosingmedium used are listed below in Table 3.

Male Sprague-Dawley rats weighing between 200-250 g were fasted for 24hours and administered ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing. A dosing group of five rats wasadministered one of the dosing solutions. For oral gavage (PO), an 11 cmRusch 8 French catheter was adapted to a 1 ml syringe with a pipettetip. The syringe was filled with dosing solution by drawing the solutionthrough the catheter, which was then wiped dry. The catheter was placeddown the esophagus leaving 1 cm of tubing past the rat's incisors.Solution was administered by pressing the syringe plunger. Forintracolonic (IC) dosing, a 7.5 cm Rusch catheter tube (French 8 or 6)was adapted to a syringe with an Eppendorf pipette tip. The syringe wasfilled with the dosing solution by drawing the solution through thecatheter tube. The catheter tube was wiped dry. K-Y jelly was applied tothe tip, avoiding contact with the eye of the tube, and the tube wasinserted into the colon through the anus until the tube was no longervisible. The solution was injected by pressing the syringe plunger, andthe tube was removed.

Blood samples were collected serially from the tail artery, typically attime=0, 15, 30, 45, 60 and 90 minutes for oral and 0, 10, 20, 30, 60 and90 minutes for IC dosing. Serum PTH concentrations were quantified by aPTH radioimmunoassay kit (Kit #RIK 6101 from Peninsula Laboratories,Inc., San Carlos, Calif.). Previous studies indicated baseline values ofabout zero. Results from the five rats in each group were averaged foreach time point. The maximum and the area under the curve (AUC) arereported below in Table 3.

TABLE 3 Oral/Intracolonic Delivery of PTH in Rats Method dosing Conju-Mean Peak of medium Volume gate PTH Serum [PTH] Conju- Adminis- (in doneDose Dose (pg/ml) gate tration water) (ml/kg) (mg/kg) (μg/kg) ±SE AUC  4PO 10% EtOH 1 100 200 86 ± 86 1285  4 PO 10% PEG300 1 100 200 39 ± 271694  4 PO 15% 1 100 200 44 ± 44 780 Kollidon 17PF  4 PO 10% PEG300 1150 200 122 ± 60  3520  4 PO 5% EtOH 1 150 200 36 ± 32 1184  4 PO 15% 1150 200 126 ± 123 3684 Kollidon 17PF  4 PO 10% PEG300 1 300 200 418 ±290 9315  4 PO 5% EtOH 1 300 200 94 ± 27 2627  4 PO 15% 1 300 200 298 ±265 6276 Killidon 17PF  4 PO 10% PEG300 1 300 200 87 ± 24 4165  4 PO 30%PEG300 1 300 200 106 ± 43  4572 14 IC 5% EtOH 0.5 100 25 501 ± 34  1633522 PO 5% citric 1 100 200 855 ± 511 27609 acid (pH = 3.85) 22 PO water 1100 200 34 ± 13 1286 (pH = 7.75) 22 PO 5% NaHCO₃ 1 100 200 225 ± 17110614 (pH = 10.13 14 PO 5% EtOH 1 250 200 320 ± 114 10933 16 PO 5% EtOH1 250 200 265 ± 47  9965 32 PO water 1 100 200 246 ± 119 4426 22 PO 15%PEG350 1 100 200 3508 ± 267  249861 22 PO 15% PG 1 100 200 2755 ± 537 242849 22 PO 15% 1 100 200 3173 ± 250  243838 Kollidon 17PF 22 PO water1 100 200 3577 ± 113  280258 22 PO 5% citric 1 100 200 946 ± 701 27117acid (pH = 2.78) 22 PO 5% NaHCO₂ 1 100 200 899 ± 730 20245 (pH = 10.04)22 PO water 1 100 200 1678 ± 763  43639 (pH = 7.82) 22 PO water 1 100200 118 ± 112 5696 22 PO 15% PEG350 1 100 200 113 ± 55  5201 22 PO 15%PG 1 100 200 273 ± 235 8193 22 PO 15% 1 100 200 126 ± 117 4993 Kollidon17PF 24 PO 5% EtOH 1 100 200 48 ± 32 2290 25 PO 5% EtOH 1 100 200 287 ±134 10486 30 PO 5% EtOH 1 100 200 0 0 49 PO 5% EtOH 1 100 200 73 ± 732785 49 PO 5% EtOH 1 100 200 1467 ± 77  106705

PTH was also administered in capsules via PO and IC routes. Mini hardgel capsules (size 9) with a total volume 25 μL and manufactured byTorpac Inc. (Fairfield, N.J., USA) were used. For IC administration, 25μg PTH/capsule and 15 mg/capsule conjugate 22 was used. For POadministration, 100 μg PTH/capsule and 20 mg/capsule conjugate 22 wasused. The powder of PTH was mixed with the oily conjugate in a vial inthe above-mentioned ratio. A clear solution resulted. Solution was addedto capsules by syringe and weighed to reach the appropriate weight.

Capsules were administered as above, with the following changes. For POdosing, a rat/hamster capsule dispenser (available from Torpac, Inc.,Fairfield, N.J.) was marked 10 cm from the dosing end. The capsule wasplaced into the dispenser, which was inserted into the mouth and downthe esophagus until the 10 cm mark meets the incisor teeth. The plungerwas pushed and withdrawn. The dosing was optionally followed byadministration of about 1.0 ml water (as in PO dosing of solutions). ForIC dosing, a rat/hamster pill dispenser was marked 7.5 cm from thedosing end. The capsule was placed into the dispenser. A small amount ofKY jelly was placed at the tip of the dispenser, and the dispenserinserted into the anus up to the 7.5 cm mark. The plunger was pressedand withdraw.

Results were obtained as above and are shown below in Table 4.

TABLE 4 Oral/Intracolonic Delivery of PTH in Rats Method dosing Conju-Mean Peak of medium gate PTH Serum [PTH] Conju- Adminis- (in Dose Dose(pg/ml) gate tration water) Dose (mg/kg) (μg/kg) ±SE AUC 22 PO capsule 120 100 2275 ± 916  242288 capsule 22 IC capsule 1 15  25 3438 ± 559  80928 capsule

Example 3 Heparin Delivery

Intracolonic Delivery

Intracolonic (IC) dosing solutions containing a conjugate and heparinsodium USP in 25% aqueous propylene glycol were prepared. Typically, theoily conjugate and powdered heparin (about 166-182 IU/mg) were dissolvedin 25% v/v aqueous propylene glycol, vortexed and placed in a sonicator(about 37° C.). The pH was adjusted to about 7 (6.5 to 8.5) with aqueousNaOH (2N). The dosing solution was sonicated to produce a clearsolution. The final volume was adjusted to 3.0 ml. The final conjugatedose, heparin dose and volume dose amounts are listed below in Table 5.

Male Sprague-Dawley rats weighing between 275-350 g were fasted for 24hours and were anesthetized with ketamine hydrochloride (88 mg/kg)intramuscularly immediately prior to dosing. A dosing group of five ratswas administered one of the dosing solutions. For intracolonic (IC)dosing, a 7.5 cm, 8 fr Rusch catheter was adapted to a 1 ml syringe witha pipette tip. The dosing catheter was inserted into the colon throughthe anus until the tube was no longer visible. The dosing solution wasexpressed slowly into the colon.

Citrated blood samples were collected by cardiac puncture following theadministration of ketamine (88 mg/kg), typically at time=0.25, 0.5, 1.0and 1.5 hours. Heparin activity was determined by utilizing theactivated partial thromboplastin time (APTT) according to the method ofHenry, J. B., Clinical Diagnosis and Management by Laboratory Methods,Philadelphia, Pa., W.B. Saunders (1979). Previous studies indicatedbaseline values of about 20 sec. Results from the five rats in eachgroup were averaged for each time point. The maximum is reported belowin Table 5.

TABLE 5 Intracolonic Delivery of Heparin Method of volume Conju- HeparinMean Peak Conju- Adminis- dose gate dose Dose APTT (sec) gate tration(ml/kg) (mg/kg) (mg/kg) ±SD 4 IC 1 50 25 163 ± 149

The above mentioned patents, applications, test methods, andpublications are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description. Allsuch obvious variations are within the full intended scope of theappended claims.

1. A polymeric delivery agent having the formula:

or a salt thereof, wherein R¹⁶ is —R³-R⁴; R³—NHC(O)NH—, —C(O)NH—,—NHC(O)—, —OOC—, —COO—, —NHC(O)O—, —OC(O)NH—, —CH₂NH—, —NHCH₂—,—CH₂NHC(O)O—, —OC(O)NHCH₂—, —CH₂NHCOCH₂O—, —OCH₂C(O)NHCH₂—,—NHC(O)CH₂O—, —OCH₂C(O)NH—, —NH—, —O—, or carbon-carbon bond; R⁴ theformula

R⁵, R⁶, R⁷, R⁸, and R⁹ are independently a bond to R³, or H, Cl, Br, F,—OH, —CH₃, —OCH₃, or —(CH₂)_(m)CH₃; R¹⁰ is a bond to R³—COOH, or—C(O)NH—R¹¹-R¹²; R¹¹ is a substituted or unsubstituted, linear orbranched alkylene having a chain length of from about 1 to about 11 or—R¹³-R¹⁴—; R¹² is a bond to R³ is —COOH, —NH₂, —OH, —C(O)—R¹⁵, —COO—R¹⁵,—NHR¹⁵, —OR¹⁵, Cl, or Br; R¹³ is a substituted or unsubstitutedphenylene; R¹⁴ is a substituted or unsubstituted, linear or branchedalkylene having a chain length of from about 1 to about 5; R¹⁵ is a bondto R³; m is from about 1 to about 4; R¹⁷—OH or —OCH₃; R²³ is H or —CH₃;and n is from about 3 to about
 200. 2. The polymeric delivery agent ofclaim 1, wherein n is from about 4 to about
 15. 3. The polymericdelivery agent of claim 1, wherein R⁹ is —OCH₃ or —OH.
 4. The polymericdelivery agent of claim 1, wherein R¹⁶ is —OOC—R⁴.
 5. The polymericdelivery agent of claim 1, wherein R¹⁷ is —OH.
 6. The polymeric deliveryagent of claim 1, wherein R¹⁷ is —OCH₃.
 7. The polymeric delivery agentof claim 1, wherein R²³ is H.
 8. The polymeric delivery agent of claim1, wherein the molecular weight of the polymer component

ranges from about 200 to about 600 daltons.
 9. The polymeric deliveryagent of claim 8, wherein the molecular weight of the polymer componentranges from about 300 to about 550 daltons.
 10. A pharmaceuticalcomposition comprising a polymeric delivery agent of claim 1 and abiologically active agent.
 11. The pharmaceutical composition of claim10, wherein the biologically active agent comprises at least oneprotein, polypeptide, peptide, hormone, polysaccharide,mucopolysaccharide, carbohydrate, or lipid.
 12. The pharmaceuticalcomposition of claim 10, wherein the biologically active agent isselected from human growth hormone, recombinant human growth hormone,bovine growth hormone, porcine growth hormone, growth hormone-releasinghormone, an interferon, α-interferon, β-interferon, γ-interferon,interleukin-1, interleukin-2, insulin, porcine insulin, bovine insulin,human insulin, human recombinant insulin, insulin-like growth factor(JGF), JGF-1, heparin, unfractionated heparin, heparinoids, dermatans,chondroitins, low molecular weight heparin, very low molecular weightheparin, ultra low molecular weight heparin, calcitonin, salmoncalcitonin, eel calcitonin, human calcitonin, erythropoietin, atrialnaturetic factor, an antigen, a monoclonal antibody, somatostatin,protease inhibitors, adrenocorticotropin, gonadotropin releasinghormone, oxytocin, leutinizing-hormone-releasing-hormone, folliclestimulating hormone, glucocerebrosidase, thrombopoietin, filgrastim,prostaglandins, cyclosporin, vasopressin, cromolyn sodium, soiumchromoglycate, disodium chromoglycate, vancomycin, parathyroid hormone,fragments of parathyroid hormone, desferrioxamine, antimicrobials,anti-fungal agents, vitamins, analogs, fragments, mimetics andpolyethylene glycol-modified derivatives of these compounds, and anycombination thereof.
 13. The pharmaceutical composition of claim 10,wherein the biologically active agent is insulin, unfractionatedheparin, low molecular weight heparin, very low molecular weightheparin, ultra low molecular weight heparin, calcitonin, parathyroidhormone, erythropoietin, human growth hormone, recombinant human growthhormone, or a combination thereof.
 14. The pharmaceutical composition ofclaim 13, wherein the biologically active agent is human growth hormone.15. The pharmaceutical composition of claim 13, wherein the biologicallyactive agent is recombinant human growth hormone.
 16. The pharmaceuticalcomposition of claim 10, wherein the pharmaceutical composition is adosage unit form and further comprises (a) an excipient, (b) a diluent,(c) a disintegrant, (d) a lubricant, (e) a plasticizer, a colorant, (g)a dosing vehicle, or (h) any combination thereof.
 17. The pharmaceuticalcomposition of claim 16, wherein the dosage unit form is a tablet, acapsule, a powder, or a liquid.
 18. A method for administering abiologically active agent to a human in need of the agent comprisingadministering to the human the pharmaceutical composition of claim 10orally, intracolonically, or intraduodenally.
 19. The method of claim18, wherein the pharmaceutical composition is administered orally. 20.The method of claim 18, wherein the pharmaceutical composition isadministered intracolonically.